The present invention relates generally to the voltage-dependent calcium channel multigene family. More particularly, the present invention relates to the characterization and isolation of an neuronal invertebrate calcium channel xcex11 subunit from Drosophila melanogaster. 
Early electrophysiological studies on invertebrate preparations revealed the presence of calcium currents and suggested the presence of multiple types of voltage-dependent calcium channels (reviewed by Hille, B., (1992), In: Ion Channels of Excitable Membranes, 2nd Ed., Sinauer, Sunderland, Mass.). Continuing studies of calcium channels have shown that they are ubiquitous since they are found in excitable cells in species ranging from Paramecium to humans. Calcium channels are involved in many cell functions including: membrane excitability, synaptic transmission, and differentiation (Tsien et al., (1988), Trends Neurosci., vol. 11, pp. 431-438). Voltage-dependent calcium channels have been studied extensively in vertebrate neuronal tissue using electrophysiological and pharmacological approaches and as a result have been divided into four classes designated L, N, T, and P (Bean, B. P., (1989), Ann. Rev. Physiol., vol. 51, pp. 367-384; Hess, P., (1990), Ann. Rev. Neurosci., vol. 13, pp. 1337-1356).
Gene cloning studies, which up to this point have focused exclusively on vertebrate species, have helped to elucidate the molecular nature of calcium channel structure and have suggested a remarkable degree of channel heterogeneity beyond that predicted from physiological and pharmacological approaches. This molecular diversity of calcium channels arises from several mechanisms. Calcium channels are comprised of multiple subunits designated xcex11 xcex12, xcex2, xcex3, and xcex4 (Catterall, W. A., (1991a), Cell, vol. 64, pp. 871-874; Catterall, W. A., (1991b), Science, vol. 253, pp.1499-1500). The (xcex12 and ∂ subunits are encoded by the same gene and are cleaved during post-translational processing whereas each of the other subunits arise from different genes. One way that calcium channel diversity arises is through the presence of a family of genes each encoding genetic variants of a given subunit. For example, in rat brain the xcex11 subunit appears to be encoded by a family of at least five different genes (Snutch et al., (1990), Proc. Natl. Acad. Sci. USA, vol. 87, pp. 3391-3395; Snutch et al., (1991), Neuron, vol. 7, pp. 45-57; Hui et al., (1991), Neuron, vol. 7, pp. 35-44; Starr et al., (1991), Proc. Natl. Acad. Sci. USA, vol. 88, pp. 5621-5625; Dubel et al., (1992) Proc. Natl. Acad. Sci. USA, vol. 89, pp. 5058-5062; Soong et al. (1993), Science, vol. 260, pp. 1133-1136). For each member the gene family further diversity is introduced by alternative splicing (Biel et al., (1990), FEBS Lett., vol. 269, pp. 409-412; Koch et al., (1990), J. Biol. Chem., vol. 265, pp. 17786-17791; Perez-Reyes et al., (1990), J. Biol. Chem., vol. 265, pp. 20430-20436; Snutch et al., (1991), Neuron, vol. 7, pp. 45-57). Recent studies point to the existence of similar molecular diversity for the other subunits as well (Williams et al., (1992a), Science, vol. 257, pp. 389-395; Williams et al., (1992b), Neuron, vol. 8, pp. 71-84). If each subunit variant can interact with more than one form of each of the other subunits to form functional channels, then there is a potential for even further molecular diversity.
Although studies of the molecular diversity of calcium channels in Drosophila are just beginning, there is evidence for structural and functional heterogeneity in this system. Binding of phenylalkylamines (calcium channel blocking agents) to Drosophila head extracts showed curvilinear Scatchard plots indicative of multiple classes differing in ligand affinity (Greenberg et al. (1989), Insect Biochem., vol. 19, 309-322). Pelzer et al., (1989), EMBO J., vol. 8, pp. 2365-2371, reported at least 8 distinct voltage-sensitive calcium channels in Drosophila head membranes following reconstitution into phospholipid bilayers. Patch clamp studies on cultured embryonic Drosophila myocytes and neurons also showed variability of channel properties, suggesting at least two types of calcium channels in Drosophila (Leung and Byerly, 1991). Further evidence for channel heterogeneity comes from differential sensitivity of Drosophila calcium channels to a purified toxin from the spider Hololena curta (Leung, H. T. and Byerly, L., (1991), J. Neurosci., vol. 11, pp. 3047-3059). This heterogeneity is further supported in another neuronal invertebrate (Periplaneta americana) where radiotracer flux studies have demonstrated the presence of dihydropyridine-insensitive and -sensitive components of phenylalkylamine-sensitive calcium uptake in nervous system and skeletal muscle membranes, respectively (Skeer et al., (1992), Insect Biochem Molec. Biol., vol. 22, pp. 539-545).
Given the heterogeneity of calcium channels in invertebrates, Drosophila provides an ideal system for a molecular genetic approach to define the significance of channel diversity by mutating individual subunit genes and determining the physiological and behavioral consequences.
Other ion channels have also been reported to date. For example, electrophysiological studies of ligand-gated ion currents in invertebrate nerve and muscle cells provide evidence for the existence of chloride channels gated by glutamate, histamine, and taurine, as well as those gated by y-aminobutyric acid (xe2x80x9cGABAxe2x80x9d) (Sattelle, D. B., (1990), Adv. Insect Physiol., vol. 22, pp. 41-56 and Lummis et al., (1990), Annu. Rev. Entomol., vol. 35, pp. 345-377). Although these findings imply the existence of a large and diverse gene family encoding ligand-gated chloride channels in invertebrates, very little is known about homologous channels of invertebrates. In French-Constant et al., (1991), Proc Natl. Acad. Sci. USA, vol. 88, pp. 7209-7213, a Drosophila melanogaster cDNA having significant predicted amino acid sequence identity to vertebrate ligand-gated chloride channel genes was isolated and mapped to a genetic locus (xe2x80x9cRdlxe2x80x9d) that confers resistance to cyclodiene insecticides and other blockers of GABA-gated chloride channels. Rdl was shown to encode a GABA subunit by the expression of functional homomultimeric GABA receptors in Xenopus oocytes following injection with RDl mRNA (French-Constant et al., (1993), Nature, vol. 363, pp. 449-451).
The only other example of a ligand-gated chloride channel gene from an invertebrate species is a GABA receptor xcex2-like subunit gene isolated from the pond snail, Lymnaea stagnalis (Harvey et al., (1991), EMBO J., vol. 10, pp. 3239-3245). The functional relationship of the product encoded by this gene to vertebrate GABA receptor xcex2 subunits was corroborated by the formation of a functional chimeric receptor with properties similar to vertebrate xcex1/xcex2 heteromultimers when the gene was co-expressed with a vertebrate a subunit in Xenopus oocytes.
The characterization and isolation of a neuronal invertebrate xcex11 calcium channel subunit gene(s) would be useful in the cloning of calcium channel subunits from other invertebrate preparations of physiological or economic importance for purposes such as screening chemical agents to identify chemical agents which specifically interact with, and bind to, the calcium channel receptor on the surface of a cell, such as, for example, organic calcium channel blocking agents, e.g., phenylalkylamines.
A major object of the present invention is the isolation and characterization of an invertebrate neuronal calcium channel xcex11 subunit gene(s).
The present invention provides for the isolation of genomic DNA fragments from Drosophila melanogaster which encode a conserved amino acid sequence unique to the voltage-dependent calcium channel multigene family. Polymerase chain reaction (xe2x80x9cPCRxe2x80x9d)-based homology and screening of cDNA libraries with homologous probe were utilized to isolate the genomic DNA fragments of the invention. Using PCR, the first neuronal invertebrate calcium channel subunit gene was cloned. That is, the neuronal calcium channel xcex11 subunit gene was cloned from Drosophila melanogaster, and designated herein as xe2x80x9cDmcalDxe2x80x9d. The CDNA clones corresponding to the DNA fragments are designated N1, W8A, SH22C, and SH22D.
The DNA sequence expressing the corresponding amino acid sequences encoding the calcium channel xcex11 subunit gene(s) of the invention can be cloned into any suitable expression vector, such as, for example, plasmid DNA, viral DNA including human viruses, animal viruses and invertebrate V4 ruses and bacteriophages to form a recombinant expression system which directs the expression of the calcium channel xcex11 subunit of the invention. It is understood that this expression system can be expressed in any suitable host cell to form a functional recombinant calcium channel receptor.
In another aspect of the invention, there is provided a method of expressing a functional neuronal invertebrate xcex11 calcium channel receptor comprising (a) transforming a host cell with the gene of the present invention e.g., gene encoding the neuronal calcium channel xcex11 subunit from Drosophila melanogaster, and (b) facilitating expression of the gene(s) in the host cell, thereby forming a functional ion channel receptor which exhibits similar pharmacological properties of calcium channel in neuronal invertebrate tissue.
In still another aspect of the invention, there is provided a method of screening a chemical agent for effectiveness as a pesticide, comprising (a) facilitating expression of the gene of the invention e.g., gene encoding the calcium channel xcex11 subunit from Drosophila melanogaster, in a host thereby forming a functional calcium channel receptor, (b) exposing the host to a chemical agent having pesticidal activity, and (c) evaluating the exposed host to determine if the functional calcium channel receptor is the target site for the pesticidal activity of the chemical agent.
In still a further aspect of the invention, there is provided a method of identifying compositions which specifically interact with, and bind to, the calcium channel receptor on the surface of a cell comprising (a) contacting a vertebrate or invertebrate cell containing the gene of the invention e.g., gene encoding the calcium channel xcex11 subunit from Drosophila melanogaster, with a plurality of chemical agents, and (b) determining those chemical agents which bind to the calcium channel expressed in the cell, thereby identifying chemical agents which specifically interact with, and bind to, the channel.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.